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Compositions and methods for detecting and identifying salmonella enterica strains

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Compositions and methods for detecting and identifying salmonella enterica strains


The present specification describes several novel SNPs of Salmonella enterica subsp. enterica. SNP profiles comprising allelic compositions at each SNP position are described which may be used to identify and differentiate different strains and serovars of Salmonella enterica subsp. enterica. The specification also describes several compositions, methods and kits useful for identifying and differentially distinguishing strains and serovars of Salmonella enterica subsp. enterica.
Related Terms: Allelic Salmonella

Browse recent Life Technologies Corporation patents - Carlsbad, CA, US
Inventors: Craig Cummings, Elena Bolchakova, Manohar Furtado
USPTO Applicaton #: #20120270216 - Class: 435 611 (USPTO) - 10/25/12 - Class 435 


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The Patent Description & Claims data below is from USPTO Patent Application 20120270216, Compositions and methods for detecting and identifying salmonella enterica strains.

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CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional Patent Application Ser. No. 61/477,142, filed Apr. 19, 2011, the entire contents of which are incorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 12, 2012, is named LT00497.txt and is 6,160,501 bytes in size.

FIELD

The present specification relates, in some embodiments, to compositions, methods and kits for detection and identification of Salmonella enterica subsp. enterica strains and serovars (serological variants). In some embodiments, the disclosure describes several novel single nucleotide polymorphisms (SNPs) and compositions derived therefrom (including probes and primers), which may be used in methods of the disclosure to detect and/or identify a Salmonella enterica subsp. enterica strain from a sample and in some embodiments to identify the serotype of a S. enterica subsp. enterica.

BACKGROUND

S. enterica strains and serovars are common food borne microbes causing diseases in humans and in animals. Some S. enterica strains cause enteric (intestinal) infections, often referred to as salmonellosis. Other Salmonella enterica strains such as Salmonella Typhi and S. Paratyphi cause typhoid fever.

Traditional serotyping, the standard method for characterization of Salmonella enterica serotypes, is laborious and time consuming, and requires the maintenance of large panels of specific antisera, which is feasible for only a small number of large microbiology reference labs.

Traditional serotyping is also unable to differentiate between evolutionarily distinct subgroups that often exist within a single polypyletic serotype.

SUMMARY

The present specification relates in some embodiments to identification of several novel single nucleotide polymorphisms (SNPs). These SNPs may be used to differentially identify S. enterica subsp. enterica strains and serovars. In some embodiments, one or more SNP's identified herein may be used to differentiate between closely related strains and serovars of Salmonella enterica subsp. enterica.

The present disclosure in some embodiments provides at least 52 SNPs operable to identify Salmonella enterica subsp. enterica strains and serovars. The fifty two novel SNPs identified herein are comprised in nucleic acid sequences comprised in SEQ ID. NOs:1-52 at position 101 in each of these sequences (see Table 2 attached at the end of the specification). The SNPs located at position 101 are shown in Table 2 by a lowercase nucleotide and correspond to a coordinate position (shown in column 1 of Table 2) in the genomic sequence of Salmonella Enteritidis (also referred to as Salmonella enterica subsp. enterica serovar Enteritidis) described in SEQ ID NO: 53 (which is also described in GenBank ID AM933172.1). SEQ ID NOs: 1-52 comprise SNPs of the disclosure at position 101 and are flanked by 100 bp of genomic DNA on either side (3′ and 5′ side) of the SNP (coordinate positions of left and right flanking sequences in reference of the S. Enteritidis genome are shown in columns 2 and 3 of Table 2).

According to some embodiments, SNPs of the disclosure may be correlated to various Salmonella enterica serotypes and strains and an SNP profile database may be created. Some embodiments describe a computer readable medium used to store a SNP profile database of the disclosure. In some embodiments, a master SNP profile database may be created having all known SNP profiles. An SNP profile of a master database will have data, such as but not limited to, the composition of an SNP allele for each SNP position, and the correlation of SNP allelic compositions at different SNP locations with a serovar and/or a strain.

Assays and methods may be designed using a SNP profile database for analysis and identification including differential identification of Salmonella strains and/or serovars. For example, to determine the strain and/or serovar, a nucleic acid isolated from a Salmonella enterica containing sample, the sample nucleic acid may be tested to determine the allelic composition for at least ten SNPs selected from a larger panel of predetermined SNPs for which serovars have been correlated (such as for example, a master SNP profile database, which in one embodiment may comprise a profile database of the 52 panel of SNPs of the disclosure). The allelic composition of each SNP tested from the sample may then be stored in a sample nucleic acid SNP profile. The sample nucleic acid SNP profile may then be compared with the master SNP profile database to determine the correlation of SNPs and SNP allelic compositions to a particular Salmonella enterica strain and/or serovar. Determining the presence of certain alleles and certain allelic compositions identifies the strain or serovar of Salmonella enterica. Comparison of SNP profiles and correlation may be performed using a computer system or may be performed manually.

In other examples, to determine the strain and/or serovar, a nucleic acid isolated from a Salmonella enterica containing sample, the sample nucleic acid may be tested to determine the allelic composition for at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine and/or at least ten SNPs selected from a larger panel of predetermined SNPs for which serovars have been correlated (such as for example, a master SNP profile database, which in one embodiment may comprise a profile database of the 52 panel of SNPs of the disclosure).

The present disclosure, in some embodiments, also provides compositions derived from the SNPs of the disclosure. Accordingly, in some embodiments, oligonucleotides comprising primers operable for amplifying (and identifying) one or more SNPs from the nucleic acid of a S. enterica strain are described. Exemplary primers comprise isolated nucleic acid sequences comprised in SEQ ID NOs: 54-105 and SEQ ID NOs: 106-157. However, primers of the disclosure are not limited to the sequences and oligonucleotides disclosed in SEQ ID NOs: 54-157, and one of skill in the art, in light of the present teachings will appreciate that additional primers are also disclosed by the present disclosure. For example, in some embodiments, isolated nucleic acid sequences of the disclosure may have at least 90% sequence identity, at least 80% sequence identity, and/or at least 70% sequence identity to nucleic acid sequences comprised in SEQ ID NOs: 54-157. Other primers may be designed that are to flank a SNP of the disclosure and to form an amplification product comprising an SNP.

In some embodiments, probes operable for identifying one or more SNPs from the nucleic acid of a S. enterica strain are provided. Exemplary probes are described in SEQ ID NOs: 158-209 which correspond to probes for one allele of a SNP, and SEQ ID NOs: 210-261, which correspond to example probes that may be used to identify the other allele of a SNP. In the probes described in SEQ ID NOs: 158-261 (see Table 3), the lowercase nucleotide corresponds to the SNP. However, probes of the disclosure are not limited to the sequences and nucleotides disclosed in SEQ ID NOs: 158-261, and one of skill in the art, in light of the present teachings will appreciate that additional probes are also disclosed by the present disclosure. For example, any nucleotide sequence having at least 10 nucleotides, at least 20 nucleotides, at least 30 nucleotides, or at least 40 nucleotides, comprising a SNP may be used as a probe. For example, in non limiting examples, any nucleotide having at least nucleotides 100-102 of sequences described in SEQ ID NOs: 1-52 and at least 10 additional nucleotides on either the 5′ or the 3′ side of these sequences may be used as a probe of the disclosure. In other non-limiting examples, any nucleotide having at least nucleotides 100-102 of sequences described in SEQ ID NOs: 1-52 and at least 5 additional nucleotides on both the 5′ and the 3′ side of these sequences may be used as a probe of the disclosure. In yet other embodiments, probe sequences of the disclosure may have at least 90% sequence identity, at least 80% sequence identity, and/or at least 70% sequence identity to nucleic acid sequences comprised in SEQ ID NOs: 54-157.

The present disclosure, in some embodiments, describes methods for identifying Salmonella enterica strains and serovars based upon determining the allelic composition of one or more SNPs identified herein. In some embodiments, a method may comprise determining the allelic composition of a panel of at least 10 SNPs to identify and/or differentially detect a strain or a serovar of S. enterica. In some embodiments, a method may comprise determining the allelic composition of all the 52 SNPs to identify and/or differentially detect a strain or a serovar of S. enterica. In some embodiments, a method may comprise determining the allelic composition of a panel of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 and/or at least 10 SNPs to identify and/or differentially detect a strain or a serovar of S. enterica.

Determining the allelic compositions and/or genotyping and/or SNP detection may comprises one or more technologies such as but not limited to sequencing (also see the next sentence), amplification, hybridization, high throughput screening methods, bead-based liquid microarray platforms, mass spectrometry, nanostring, microfluidics, chemiluminescence, oligonucleotide ligation, enzyme technologies and combinations thereof. Determining the allelic compositions and/or genotyping and/or SNP detection by sequencing may comprises one or more technologies and/or platforms such as but not limited to genomic sequencing, sequencing targeted regions on CE or semiconductor platforms, multiplex sequencing of all SNP containing regions by CE or semiconductor sequencing and/or by combining with sequencing regions such as but not limited to rfb, fliC and fljB regions on the same platform. Molecular assays to detect SNPs may include amplification performed by a variety of methods such as but not limited to TaqMan®, SnapShot® and other high throughput screening methods know in the art in light of this specification.

Some methods for identifying and/or detecting S. enterica strains and/or serovars in a sample may comprise using an isolated nucleotide sequence composition of the disclosure for detection. Exemplary compositions of the disclosure used for detection methods may comprise, but are not limited to, SEQ ID NO: 54-157, and/or SEQ ID NO:158-261, fragments thereof, at least 10 contiguous nucleotide sequences thereof, complements thereof, isolated nucleic acid sequence comprising at least 90% nucleic acid sequence identity to the sequences set forth above and/or labeled derivatives thereof.

In some embodiments, methods of the disclosure may comprise: isolating a nucleic acid from a sample suspected of having a S. enterica strain and/or a sample from which one desires to detect and/or identify a specific S. enterica strain; amplifying one or more SNP comprising nucleic acid sequences (target nucleic acid sequence) from the nucleic acid from the sample to form an amplification product; and determining the allelic composition of the SNP comprised in the amplified product. Amplification may be repeated using a different set of primer pairs, each primer pair operable to hybridize to and amplify a target nucleic acid sequence comprising another SNP and determining the allelic composition of each SNP until the allelic composition of a panel of SNPs is determined. Once sufficient allelic composition is determined a correlation may be made to which serovar or strain of S. enterica the allelic composition may be assigned to. In some embodiments, at least 10 SNPs allelic compositions may be determined. In some embodiments, a panel of at least 10 SNPs selected from the SNPs comprised in SEQ ID NOs: 1-52 may be amplified and detected. In some embodiments, all SNPs comprised SEQ ID NOs: 1-52 may be amplified and detected to identify and/or type a strain of S. enterica. Amplification reactions may be multiplexed to detect a panel of SNPs.

In some embodiments, a pair of primers used for amplification may comprise the nucleic acid sequence of SEQ ID NO: 54-105, and/or SEQ ID NO: 106-157 and/or labeled derivatives thereof. For example, as shown in Table 3, a primer pair shown as reverse and forward primers may be used to amplify the corresponding SNP in the same row. Thus for example, a primer pair may comprise a first primer and a second primer, the first primer having a SEQ ID NO: 54 may be used as a forward primer, the second primer having a nucleic acid of SEQ ID NO: 106 may be used as a reverse primer may be used to amplify a SNP comprised in SEQ ID NO 1. Primers may be labeled and nucleic acid amplification products may be detected and/or identified by a variety of methods known in the art including but not limited to size analysis of an amplified product; sequencing an amplified product; hybridization with a probe; 5′ nuclease digestion; single-stranded conformation polymorphism; allele specific hybridization; primer specific extension; and oligonucleotide ligation assay.

In some embodiments, methods of the disclosure may comprise: isolating a nucleic acid from a sample suspected of having a S. enterica strain and/or a sample from which one desires to detect and/or identify a specific S. enterica strain; hybridizing one or more SNP comprising regions of the nucleic acid from the sample using one or more probes, each probe designed to bind specifically to a region comprising an SNP; and detecting the hybridized probe-nucleic acid complex. Probes used may be labeled to enable detection. Multiplex hybrid detection maybe enable by using differentially labeled probes. Other detection methods may comprise size analysis of the hybridized product; sequencing an amplified product; hybridization with a probe; 5′ nuclease digestion; single-stranded conformation polymorphism; and/or allele specific hybridization.

In some embodiments, probes used for hybridization and/or for detection of amplified products comprising a SNP may comprise the nucleic acid sequence of SEQ ID NO: 158-209, and/or SEQ ID NO: 210-261, and may comprise labeled derivatives thereof. For example, as shown in Table 3, probes 1 labeled with FAM-MGB and/or probes 2 labeled with VIC-MGB may be used to identify the corresponding SNP in the same S. Entritidis coordinate position (see Tables 2 and 3). Thus for example, probes having SEQ ID NO: 158 and SEQ ID NO: 210 may be used to hybridize to an SNP comprised in SEQ ID NO: 1. Each probe is operable to hybridize to one allelic composition of the SNP described in SEQ ID. NO: 1. For example, probe having SEQ ID NO: 158 has a “g” (guanine) at the complementary position, hence it is operable to selectively hybridize to an allelic variant of the SNP in SEQ ID NO: 1 having a “c” (cytosine) allelic composition, whereas probe having SEQ ID NO: 210 has an “a” (adenine) at the complementary position, hence it is operable to selectively hybridize to an allelic variant of the SNP in SEQ ID NO: 1 having a “t” allelic composition. In some embodiments, both probes may be used to determine what the allelic composition of the SNP in SEQ ID NO: 1. For example, a first probe labeled with a first label may be used to hybridize to one allele of the SNP (of SEQ ID NO. 1 for example having the “c” allelic composition) and a second probe labeled with a second label may be used to hybridize to the other allele of a SNP (of SEQ ID NO: 1, this may be for example the “t” allelic composition. In this example embodiment of SEQ ID NO. 1, if a first probe of SEQ ID NO: 158 and a second probe of SEQ ID NO: 210 are used and only the FAM-MGB signal is detected, the allelic composition of the SNP in SEQ ID NO: 1 is “c.” If however, only the VIC-MGB signal is detected, the SNP allelic composition of the SNP of SEQ ID NO: 1 is “t.”

In some embodiments, a panel of at least 5 and/or at least 10 SNPs selected from the SNPs comprised in SEQ ID NOs: 1-52 may be amplified and the composition of the SNP determined. In some embodiments, all SNPs comprised SEQ ID NOs: 1-52 may be amplified and the allelic composition determined to identify and/or type a strain of S. enterica.

Molecular assays to detect SNPs may include amplification performed by a variety of methods such as but not limited to TaqMan®, SnapShot® and other high throughput screening methods know in the art in light of this specification.

Methods of the disclosure may also comprise determining the allelic compositions (i.e. genotyping and/or SNP detection) by one or more technologies in addition to amplification such as but not limited to sequencing, hybridization, hybridization on bead-based liquid microarray platforms, high throughput screening methods, mass spectrometry, nanostring, microfluidics, chemiluminescence, oligonucleotide ligation, enzyme technologies and combinations thereof. In methods of the disclosure, determining the allelic compositions by sequencing may comprises one or more technologies and/or platforms such as but not limited to genomic sequencing, sequencing targeted regions on CE or semiconductor platforms, multiplex sequencing of all SNP containing regions by CE or semiconductor sequencing and/or by combining with sequencing regions such as, but not limited to, rfb, fliC and fljB regions on the same platform.

Methods of the disclosure may provide one or more advantages listed here. For example, in some embodiments, the methods provide a molecular assay in contrast to traditional immunoassays which may be easier, and/or faster, and/or allow for portable testing options, and/or that may be performed in more accessible settings than traditional serotyping methods.

Some embodiments of the present disclosure provide kits for detection and/or differential detection and/or identification of S enetrica strains. A kit of the disclosure may comprise one or more isolated nucleic acid sequences of the disclosure as set forth herein. Some nucleic acid compositions of the disclosure may comprise primers for amplification of target nucleic acid sequences comprising one or more SNPs that are specific to one or more strains of a S. enterica that may be present in a sample. Some nucleic acid compositions of the disclosure may comprise probes for the detection of target nucleic acid sequences and/or amplified target nucleic acid regions comprising one or more SNPs from a S. enterica strain present in a sample. Probes and primers comprised in kits may be labeled. Kits may additionally comprise one or more components such as, but not limited to: buffers, enzymes, nucleotides, salts, reagents to process and prepare samples (e.g., reagents to isolate nucleic acids), probes, primers, agents to enable detection and control nucleotides. Each component of a kit of the disclosure may be packaged individually or together in various combinations in one or more suitable container means.

While specific advantages have been disclosed hereinabove, it will be understood that various embodiments may include all, some, or none of the previously disclosed advantages. Other technical advantages may become readily apparent to those skilled in the art in light of the teachings of the present disclosure.

These and other features of the present teachings will become more apparent from the detailed description in sections below.

BRIEF DESCRIPTION OF THE DRAWINGS

One or more embodiments of the present disclosure may be better understood in reference to one or more the drawings below. The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

FIG. 1 depicts hierarchical clustering of SNP profiles in Salmonella enterica strains, according to one embodiment of the disclosure.

DETAILED DESCRIPTION

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to limit the scope of the current teachings. In this application, the use of the singular includes the plural unless specifically stated otherwise. Also, the use of “comprise”, “contain”, and “include”, or modifications of those root words, for example but not limited to, “comprises”, “contained”, and “including”, are not intended to be limiting. Use of “or” means “and/or” unless stated otherwise. The term “and/or” means that the terms before and after can be taken together or separately. For illustration purposes, but not as a limitation, “X and/or Y” can mean “X” or “Y” or “X and Y”.

Whenever a range of values is provided herein, the range is meant to include the starting value and the ending value and any value or value range there between unless otherwise specifically stated. For example, “from 0.2 to 0.5” means 0.2, 0.3, 0.4, 0.5; ranges there between such as 0.2-0.3, 0.3-0.4, 0.2-0.4; increments there between such as 0.25, 0.35, 0.225, 0.335, 0.49; increment ranges there between such as 0.26-0.39; and the like.

The term “cells” refers to the smallest structural unit of an organism that is capable of independent functioning, consisting of one or more nuclei, cytoplasm, and various organelles, all surrounded by a semipermeable cell membrane.

As used herein, the term “contacting” as used herein refers to bringing in contact at least two moieties (reagents, cells, nucleic acids) to bring about a change or a reaction in one or all the moieties. The process of contacting may also comprise “incubating” (contacting for a certain time lengths) and/or incubating at certain temperatures to bring about the change or reaction. In some embodiments “contacting” may also refers to the hybridization between a primer and its substantially complementary region.

The terms “ambient conditions” and “room temperature” are interchangeable and refer to common, prevailing, and uncontrolled atmospheric and weather conditions in a room or place.

As used herein, the term “analyzing” refers to evaluating and comparing the results of a method. In some exemplary embodiments, “analyzing” refers to evaluating and comparing the results of a sample tested to a second sample and/or to a control in a method of the disclosure.

As used herein, “complement” and “complements” are used interchangeably and refer to the ability of a nucleotide, a polynucleotide or two single stranded polynucleotides (for instance, a primer and a target polynucleotide) to base pair with each other, where an adenine on one strand of a polynucleotide will base pair to a thymine or uracil on a strand of a second polynucleotide and a cytosine on one strand of a polynucleotide will base pair to a guanine on a strand of a second polynucleotide. Two polynucleotides are complementary to each other when a nucleotide sequence in one polynucleotide can base pair with a nucleotide sequence in a second polynucleotide. For instance, 5′-ATGC-3′ and 5′-GCAT-3′ are complementary.

As used herein the term “complementary nucleotide sequence” and “complementary sequences” refers to a (second) nucleotide sequence which, by base pairing, is the complement of a first nucleotide sequence. For example, a forward strand with the sequence 5′-ATGGC-3′ would have the complementary nucleotide sequence 3′-TACCG-5′, also termed the “reverse strand.”

The terms “detecting” and “detection” and “determining” are used in a broad sense herein and encompass any technique by which one can determine the absence or presence of something, and/or identify a nucleic acid sequence and/or an exact allelic composition at an SNP locus that may have one or more alleles at that location and/or a protein encoded by a nucleic acid sequence. In some embodiments, detecting comprises quantitating a detectable signal from the nucleic acid, including without limitation, a real-time detection method, such as quantitative PCR (“Q-PCR”), detection of labels. In some embodiments, detecting comprises determining the sequence of an amplification product to determine the sequence of an allele.

As used here, “distinguishing” and “distinguishable” are used interchangeably and refer to differentiating between at least two results from substantially similar or identical reactions, including but not limited to, two different amplification products, two different melting temperatures, two different melt curves, and the like. The results can be from a single reaction, two reactions conducted in parallel, two reactions conducted independently, i.e., separate days, operators, laboratories, and so on. As used herein, “presence” refers to the existence (and therefore to the detection) of a reaction, a product of a method or a process (including but not limited to, an amplification product resulting from an amplification reaction), or to the “presence” and “detection” of an organism such as a pathogenic organism or a particular strain or species of an organism.

The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

As used herein, the term “genome” refers to the complete nucleic acid sequence, containing the entire genetic information, of a bacterium, a virus, a plasmid, a gamete, an individual, a population, a species, or a strain of a species.

As used herein, the term “pseudochromosome” refers to the concatenation, in their most likely order, of all available sequence contigs and scaffolds derived from sequencing of a bacterial genome, in which undefined gaps between contigs and scaffolds are represented by unidentified nucleobases.

As used herein, the term “genomic DNA” refers to the chromosomal DNA sequence of a gene or segment of a gene including the DNA sequence of non-coding as well as coding regions. Genomic DNA also refers to DNA isolated directly from cells, chromosomes or plasmid(s) within the genome of an organism, or cloned copies of all or part of such DNA.

Identification and Selection of SNPs

The present specification relates in some embodiments to identification and selection of several novel single nucleotide polymorphisms (SNPs) that may be used to differentially identify S enterica strains from each other including in some embodiments, to differentiate between closely related strains of Salmonella.

A “single nucleotide polymorphism” or “SNP” refers to a variation in the nucleotide sequence of a polynucleotide that differs from another polynucleotide by a single nucleotide difference. For example, without limitation, exchanging one A for one C, G or T in the entire sequence of polynucleotide constitutes a SNP. It is possible to have more than one SNP in a particular polynucleotide. For example, at one position in a polynucleotide, a C may be exchanged for a T, at another position a G may be exchanged for an A and so on. When referring to SNPs, the polynucleotide is most often DNA.

The present disclosure in some embodiments provides at least 52 SNPs comprised in nucleic acid sequences comprised in SEQ ID. NOs: 1-52 at position 101 in each of these sequences. SEQ ID NOs: 1-52 comprise SNPs of the disclosure at position 101 and are flanked by 100 bp of genomic DNA on either side (3′ and 5′ side) of the SNP.

The SNP position in SEQ ID. NOs: 1-52 is indicated by a lowercase letter. The flanking sequences and SNPs are corresponding sequences from a single Salmonella genome (S. Enteritidis, GenBank ID AM933172.1, also described herein as SEQ ID NO. 53). Other Salmonella genomes may differ slightly at other bases within this sequence.

In some embodiments, the disclosure describes compositions comprising isolated nucleic acid sequences having SEQ ID. NOs: 1-52, fragments thereof (including fragments having at least 10 contiguous nucleotides thereof, fragments having at least 20 contiguous nucleotides thereof, fragments having at least 30 contiguous nucleotides thereof, fragments having at least 40 contiguous nucleotides thereof, fragments having at least 50 contiguous nucleotides thereof, and/or fragments having at least 60 contiguous nucleotides thereof), as well as sequences having at least 90% sequence identity, at least 80% sequence identity, and/or at least 70% sequence identity to nucleic acid sequences comprised therein and complementary sequences thereof.

In some embodiments, isolated nucleic acid sequences of the disclosure comprising SEQ ID. NOs: 1-52 may have at least 90% sequence identity, at least 80% sequence identity, and/or at least 70% sequence identity to nucleic acid sequences comprised therein.

An isolated SNP-containing nucleic acid molecule may comprise one or more SNP positions disclosed by the present invention with flanking nucleotide sequences on either side of the SNP positions. A flanking sequence can include nucleotide residues that are naturally associated with the SNP site and/or heterologous nucleotide sequences. Although sequences described have 100 bp of flanking nucleic acid sequences, the flanking sequence may be up to about 500, 400, 300, 200, 100, 60, 50, 40, 30, 25, 20, 15, 10, 8, or 4 nucleotides (or any other length in-between) on either side of a SNP position.

In some embodiments, the present disclosure relates to identification of novel SNP loci in Salmonella enterica strains. In order to identify nucleic acid sequences that are conserved in all Salmonella enterica strains, 34 completely sequenced S. enterica serotypes, including 16 presently sequenced strains and 18 publicly available serotypes, were aligned to the S. Enteritidis genome using the open-source whole-genome alignment tool, MUMmer. A list of 34 Salmonella enterica strains are provided in Table 1 attached at the end of the specification. A total of 282 kb of conserved chromosomal sequences were identified. Each of the genomes was then compared to the S. Enteritidis genome (GenBank ID AM933172.1 represented by SEQ ID NO. 53) and 10,101 single nucleotide polymorphisms (SNPs) in these conserved chromosomal regions were identified.

The 10,101 SNPs were screened to identify SNPs specific for different strains. In order to select the most highly discriminative SNPs, the set of 34 genomes was randomly partitioned into two groups 10,000 times, and at each iteration, an SNP with the most highly correlated profile (i.e., most capable of distinguishing between the two random groups) was selected. These were then sorted to obtain the best set of 48 SNPs (shown in Table 2 as SEQ ID NOs: 1-3, 6-13, 15-45, and 47-52. These 48 SNPs identified in the present disclosure vary significantly across the population, but are not associated with any phylogenetic signal. In some embodiments, the 48 SNPs are functional to discriminate between most strains of Salmonella enterica subsp. enterica.

In some embodiments, the disclosure also identifies four additional SNP\'s operable to differentially identify and discriminate between closely related strains of S. enterica, including between S. Enteritidis and S. Gallinarum; between S. Paratyphi C; and S. Choleraesuis; and between S. Johannesburg and S. Urbana. These four additional SNPs are described in Table 2 as SEQ ID NOs: 4, 5, 14, and 46. These 4 SNPs were manually selected by the present inventors using the selection criteria described above.

Accordingly, the present disclosure identifies a total of 52 unique SNP sequences that are listed in Table 2 as having SEQ ID NOs: 1-52. In some embodiments, the 52 SNPs or subsets selected therefrom are described as an SNP panel. For example an SNP panel of the disclosure may comprise at least 10 SNPs selected from the 52 SNPs identified.

In some embodiments, a SNP panel of the disclosure may be stored in a database such as a database located in a computer readable medium. “Computer readable media” refers to any media which can be read and accessed directly by a computer, and includes, but is not limited to: magnetic storage media such as floppy discs, hard storage medium and magnetic tape; optical storage media such as optical discs or CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories, such as magnetic/optical media. By providing such computer readable media, a database comprising SNPs or other data compiled on SNPs may be routinely accessed by a user and used for analysis or designing experiments.

In some embodiments, SNPs of the disclosure may be correlated to Salmonella serotypes and/or strains and an SNP profile database may be created. Some embodiments describe a computer readable medium used to store a SNP profile database of the disclosure.

In some embodiments, SNP profile database and SNP panel databases may be used to design and analyze assays and methods that use a computer system for analysis and identification of Salmonella strains and/or serovars (including differential identification assays and assays correlating SNPs with serotypes). Such methods may also involve data analysis using a “data analysis module” which may include any person or machine, individually or working together, which analyzes the sample and determines the genetic information contained therein. The term may include a person or machine within a laboratory setting.

A “computer system” refers to the hardware means, software means and data storage means used to compile the data of the present invention. The minimum hard ware means of computer-based systems of the invention may comprise a central processing unit (CPU), input means, output means, and data storage means. Desirably, a monitor is provided to visualize structure data. The data storage means may be RAM or other means for accessing computer readable media of the invention. Examples of such systems are microcomputer workstations available from Silicon Graphics Incorporated and Sun Microsystems running Unix based, Linux, Windows NT, XP or IBM OS/2 operating systems.

Hierarchical clustering of SNP profiles (depicted in FIG. 1) showed that, in some embodiments, strains of the same serotype have identical profiles. For example, each of four S. Typhimurium strains, each of two S. Typhi strains and each of two S. Paratyphi A strains had identical SNP profiles. The closely related pairs (listed above) differ by two SNPs. Other more unrelated strains differ significantly. For example, the next closely matched pair was the S. Minnesota and the S. Gaminara pair which differed by eight SNPs.

Validation of SNP Loci

The genetic loci of SNPs identified in the present disclosure were tested and analyzed in several additional strains of S. enterica (including 13 newly available S. enterica genomes—see details below). The 52 SNP loci were found to be present in every strain of S. enterica analyzed by the present inventors.

For example, in order to test the effectiveness of the 52 SNP panel to distinguish new serovars, the genotype at each of the SNP positions was extracted from an additional 13 publicly available draft S. enterica genomes, bringing the total number of genomes analyzed to 47 strains representing 37 serovars. Most new serovars were clearly distinguishable from the set of serovars used to construct the 52 SNP panel. The only exception was serovar 4,5,12:i:-, which could not be differentiated from S. Typhimurium at these SNP positions. These two serovars are known to be very closely related.

Two of the draft genomes are additional isolates of serovars used to construct the panel and comprise the serovars Heidelberg and Schwarzengrund. These have identical profiles to the genomes of completely sequenced Salmonella genomes representing the same serovar. In addition, the two draft S. Kentucky genomes were also identical.

Two of the 13 new serovars tested, including the draft genomes of S. Newport and S. Saintpaul, gave discrepant results. The draft S. Newport genome was found to have a genome profile completely unrelated to the complete Newport genome that used to construct the 52 SNP profile of the present disclosure. Previous work, based on MLST, has demonstrated that Newport isolates can be separated into two distinct evolutionary lineages. Based on the present results it appears that the draft Newport genome represents the other evolutionary lineage. The two draft S. Saintpaul genomes also gave very different profiles. However, as stated at the NCBI genome project pages, “The selected strains are from separate lineages representing genovar groupings: strain SARA23 falls within the main clade for the serovar, and strain SARA29 is an outlier.” Together, the draft S. Newport and S. Saintpaul results indicate that a single serovar may comprise multiple unrelated genetic types, and that these will be reflected in different SNP profiles.

These results further reinforce the stability of the present 52 SNP profile for serovar identification. Accordingly, the present disclosure provides a SNP profile that may be used to establish an interpretable SNP profile for any Salmonella enterica strain using a molecular assay format using the same set-of assay reagents.

In contrast to the present SNPs and SNP profile based assays, another previous SNP based assay targeted a set of five S. enterica serovars. These assays are however limited to be able to identify only the five targeted serovars and do not provide identification of non-targeted serovars (Ben-Darif, JOURNAL OF CLINICAL MICROBIOLOGY, April 2010, p. 1055-1060 Vol. 48, No. 4).

Compositions of the Disclosure

The present disclosure, in some embodiments, also provides compositions derived from one or more SNPs identifies here. Accordingly, in some embodiments, oligonucleotides comprising primers operable for amplifying (and identifying) one or more SNPs from the nucleic acid of a S. enterica strain are described. Exemplary primers may comprise isolated nucleic acid sequences comprised in SEQ ID NOs: 54-105 and SEQ ID NOs: 106-157. However, primers of the disclosure are not limited to the sequences and oligonucleotides disclosed in SEQ ID NOs: 54-157, and one of skill in the art, in light of the present teachings will appreciate that additional primers are also disclosed by the present disclosure. For example, in some embodiments, isolated nucleic acid sequences of the disclosure may have at least 90% sequence identity, at least 80% sequence identity, and/or at least 70% sequence identity to nucleic acid sequences comprised in SEQ ID NOs: 54-157. In other examples, any nucleotide having at least 10 nucleotides on one strand and at least 10 nucleotides on a complementary strand of the first strand that flank a SNP of the disclosure to form an amplification product.

In some embodiments, compositions of the disclosure comprise probes operable for identifying one or more SNPs from the nucleic acid of a S. enterica strain are provided. Exemplary probes are described in SEQ ID NOs: 158-209 which correspond to probes for one allele of an SNP, and SEQ ID NOs: 210-261 correspond to example probes that may be used to identify an SNP on the other allele. In the probes described in SEQ ID NOs: 158-261 (see Table 3), the lowercase nucleotide corresponds to the SNP. However, probes of the disclosure are not limited to the sequences and nucleotides disclosed in SEQ ID NOs: 158-261, and one of skill in the art, in light of the present teachings will appreciate that additional probes are also disclosed by the present disclosure. For example, any nucleotide sequence having at least 10 nucleotides, at least 20 nucleotides, at least 30 nucleotides, or at least 40 nucleotides, comprising a SNP may be used as a probe. For example, in non limiting examples, any nucleotide having at least nucleotides 100-102 of sequences described in SEQ ID NOs: 1-52 and at least 10 additional nucleotides on either the 5′ or the 3′ side of these sequences may be used as a probe of the disclosure. In other non-limiting examples, any nucleotide having at least nucleotides 100-102 of sequences described in SEQ ID NOs: 1-52 and at least 5 additional nucleotides on both the 5′ and the 3′ side of these sequences may be used as a probe of the disclosure. In yet other embodiments, primer sequences of the disclosure may have at least 90% sequence identity, at least 80% sequence identity, and/or at least 70% sequence identity to nucleic acid sequences comprised in SEQ ID NOs: 54-157.

In some embodiments, the disclosure describes compositions comprising isolated nucleic acid sequences having SEQ ID. NOs: 1-52, fragments thereof (including fragments having at least 10 contiguous nucleotides thereof, fragments having at least 15 contiguous nucleotides thereof, fragments having at least 20 contiguous nucleotides thereof, fragments having at least 30 contiguous nucleotides thereof, fragments having at least 40 contiguous nucleotides thereof, fragments having at least 50 contiguous nucleotides thereof, and/or fragments having at least 60 contiguous nucleotides thereof), as well as sequences having at least 90% sequence identity, at least 80% sequence identity, and/or at least 70% sequence identity to nucleic acid sequences comprised therein and complementary sequences thereof. In some embodiments, fragments of the isolated nucleotide sequences derived from SEQ ID NO: 1-SEQ ID NO: 52, as described above, also comprise at least nucleotides located at positions 100-103 of SEQ ID NO: 1-SEQ ID NO: 52, i.e., comprise a SNP of the disclosure. Isolated nucleic acids fragments comprising at least 10 (or more) contiguous nucleotides may be used as primers and/or probes of the disclosure. In some embodiments, isolated nucleic acids fragments comprising at least 10, or at least 15, (or more) contiguous nucleotides and further comprising at least nucleotides located at positions 100-103 of SEQ ID NO: 1-SEQ ID NO: 52, i.e., comprise a SNP of the disclosure may be used as primers and/or probes of the disclosure.

In some embodiments, isolated nucleic acid sequences of the disclosure comprising SEQ ID. NOs: 1-52 may have at least 90% sequence identity, at least 80% sequence identity, and/or at least 70% sequence identity to nucleic acid sequences comprised therein.

Nucleic acids, probes and primers of the disclosure may be further labeled. The term “label” refers to any moiety which can be attached to a molecule and: (i) provides a detectable signal; (ii) interacts with a second label to modify the detectable signal provided by the second label, e.g. FRET; (iii) stabilizes hybridization, i.e. duplex formation; or (iv) provides a capture moiety, i.e. affinity, antibody/antigen, ionic complexation. Labeling can be accomplished using any one of a large number of known techniques employing known labels, linkages, linking groups, reagents, reaction conditions, and analysis and purification methods. Labels include light-emitting compounds which generate a detectable signal by fluorescence, chemiluminescence, or bioluminescence (Kricka, L. in Nonisotopic DNA Probe Techniques (1992), Academic Press, San Diego, pp. 3-28). Another class of labels comprise hybridization-stabilizing moieties which serve to enhance, stabilize, or influence hybridization of duplexes, e.g. intercalators, minor-groove binders, and cross-linking functional groups (Blackburn, G. and Gait, M. Eds. “DNA and RNA structure” in Nucleic Acids in Chemistry and Biology, 2nd Edition, (1996) Oxford University Press, pp. 15-81). Yet another class of labels effect the separation or immobilization of a molecule by specific or non-specific capture, for example biotin, digoxigenin, and other haptens (Andrus, A. “Chemical methods for 5′ non-isotopic labeling of PCR probes and primers” (1995) in PCR 2: A Practical Approach, Oxford University Press, Oxford, pp. 39-54). A label may include but is not limited to a dye, a radioactive isotope, a chemiluminescent label, a fluorescent moiety, a bioluminescent moiety, and/or an enzyme.

As used herein, the terms “polynucleotide”, “oligonucleotide”, and “nucleic acid sequences” are used interchangeably and refer to single-stranded and double-stranded polymers of nucleotide monomers, including without limitation 2′-deoxyribonucleotides (DNA) and ribonucleotides (RNA) linked by internucleotide phosphodiester bond linkages, or internucleotide analogs, and associated counter ions, e.g., H+, NH4+, trialkylammonium, Mg2+, Na+, and the like. A polynucleotide may be composed entirely of deoxyribonucleotides, entirely of ribonucleotides, or chimeric mixtures thereof and can include nucleotide analogs. The nucleotide monomer units may comprise any nucleotide or nucleotide analog. Polynucleotides typically range in size from a few monomeric units, e.g. 5-40 when they are sometimes referred to in the art as oligonucleotides, to several thousands of monomeric nucleotide units. Unless denoted otherwise, whenever a polynucleotide sequence is represented, it will be understood that the nucleotides are in 5′ to 3′ order from left to right and that “A” denotes deoxyadenosine, “C” denotes deoxycytosine, “G” denotes deoxyguanosine, “T” denotes thymidine, and “U” denotes deoxyuridine, unless otherwise noted.

An “isolated” polynucleotide or oligonucleotide is one that is substantially pure of the materials with which it is associated in its native environment. By substantially free, is meant at least 50%, at least 55%, at least 60%, at least 65%, at advantageously at least 70%, at least 75%, more advantageously at least 80%, at least 85%, even more advantageously at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, most advantageously at least 98%, at least 99%, at least 99.5%, at least 99.9% free of these materials.

An “isolated” nucleic acid molecule is a nucleic acid molecule separate and discrete from the whole organism with which the molecule is found in nature; or a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences in association therewith.

As used herein, the term “nucleotide” or “nt” refers to a base-sugar-phosphate combination. Nucleotides are monomeric units of a nucleic acid molecule (DNA and RNA). The term nucleotide includes ribonucleoside triphosphates ATP, UTP, CTG, GTP and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives include, for example, 7-deaza-dGTP and 7-deaza-dATP. The term nucleotide as used herein also refers to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Examples of dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP.

As used herein, the phrase “nucleic acid molecule” refers to a sequence of contiguous nucleotides (riboNTPs, dNTPs or ddNTPs, or combinations thereof) of any length which can encode a full length polypeptide or a fragment of any length thereof, or which can be non-coding. As used herein, the terms “nucleic acid molecule” and “polynucleotide” can be used interchangeably and include both RNA and DNA.

The terms “identity”, “nucleic acid sequence identity” and “sequence identity” are used interchangeably and refer to the percentage of pair-wise identical residues—following homology alignment of a sequence of a polynucleotide with a sequence in question—with respect to the number of residues in the longer of these two sequences. The term “identity” as known in the art refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between nucleic acid molecules or polypeptides, as the case may be, as determined by the match between strings of two or more nucleotide or two or more amino acid sequences. “Identity” measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., “algorithms”).

The term “percent (%) nucleic acid sequence identity” with respect to a nucleic acid sequence refers to the percentage of nucleotides in a first sequence that are identical with the nucleotides in a second nucleic acid sequence of interest, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are known to one of skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.

Percent nucleic acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program may be downloaded from http://www.ncbi.nlm.nih.gov or otherwise obtained from the National Institute of Health, Bethesda, Md. NCBI-BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask=yes, strand=all, expected occurrences=10, minimum low complexity length=15/5, multi-pass e-value=0.01, constant for multi-pass=25, dropoff for final gapped alignment=25 and scoring matrix=BLOSUM62.

In situations where NCBI-BLAST2 is employed for sequence comparisons, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows: 100 times the fraction W/Z where W is the number of nucleotides scored as identical matches by the sequence alignment program NCBI-BLAST2 in that program\'s alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C.

Methods Using a SNP Panel of the Disclosure


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stats Patent Info
Application #
US 20120270216 A1
Publish Date
10/25/2012
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File Date
12/20/2014
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